Collagen serves as a structural scaffold and a barrier between tissues, and thus collagen catabolism (collagenolysis) is required to be a tightly regulated process in normal physiology. In turn, the destruction or damage of collagen during pathological states plays a role in tumor growth and invasion, cartilage degradation, or atherosclerotic plaque formation and rupture. Only a small number of proteases have been identified capable of efficient processing of triple-helical regions of collagens. Several members of the zinc metalloenzyme family, specifically matrix metalloproteinases (MMPs), possess collagenolytic activity. A mechanistic understanding of the cleavage of intact collagens has been pursued for many years;the results of such studies could lead to the development of truly selective MMP inhibitors. Our laboratory developed triple-helical peptides (THPs) as MMP substrates, with the goal of using these models to dissect collagenolytic behavior and develop selective MMP inhibitors. In the most recent funding period of the present project (04/01/08-present), we have experimentally derived the initial steps of collagenolysis and identified specific residues involved in this process, quantified the roles of specific collagen residues in MMP substrate specificity, and developed selective MMP inhibitors based on secondary binding sites (exosites), and demonstrated the in vivo use of such inhibitors. Our studies also revealed intriguing, unexpected behaviors of collagenolytic MMPs, such as MMPs bind the triple-helix using slightly different orientations, one can use exosite binding THPs to inhibit some, but not all, proteolytic activities for a given enzyme, which could significantly reduce side effects, and substrate selectivity seen for single-stranded peptides is not observed in the triple-helix. The research plan described herein focuses on further development of triple-helical probes for teasing out collagenolytic MMP sequence specificities, identifying selective MMP inhibitors, and advancing the mechanism of collagenolysis. To achieve these goals we propose to (a) identify THP sequence preferences for the MMP family utilizing positional scanning combinatorial libraries, (b) design and characterize MMP selective, homotrimeric and heterotrimeric triple-helical transition-state analog inhibitors, and (c) explore the latter steps of collagenolysis usin state-of-the-art NMR spectroscopic techniques and design exosite binding THPs. Select inhibitors will be tested in mouse models of breast carcinoma and melanoma. Ultimately, we would like to obtain inhibitors that target those proteases implicated in cancers such as melanoma and breast carcinoma (MMP-1, MMP-2, MMP-9, MMP-13, and MT1-MMP) while sparing proteases with host-beneficial functions (MMP-3 and MMP-8).

Public Health Relevance

The present study is designed to create a novel class of therapeutic agents to selectively stop the action of tumor-associated enzymes that degrade proteins (proteases). These proteases have been shown to be important for cancer progression, and thus blocking their function will impair the spread of cancer.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
2R01CA098799-12
Application #
8506471
Study Section
Drug Discovery and Molecular Pharmacology Study Section (DMP)
Program Officer
Knowlton, John R
Project Start
2003-03-05
Project End
2018-03-31
Budget Start
2013-04-01
Budget End
2014-03-31
Support Year
12
Fiscal Year
2013
Total Cost
$335,644
Indirect Cost
$123,671
Name
Torrey Pines Institute for Molecular Studies
Department
Type
DUNS #
605758754
City
Port Saint Lucie
State
FL
Country
United States
Zip Code
34987
Marcink, Tara C; Simoncic, Jayce A; An, Bo et al. (2018) MT1-MMP Binds Membranes by Opposite Tips of Its ? Propeller to Position It for Pericellular Proteolysis. Structure :
Stawikowski, Maciej J; Fields, Gregg B (2018) Tricine as a convenient scaffold for the synthesis of C-terminally branched collagen-model peptides. Tetrahedron Lett 59:130-134
Tokmina-Roszyk, Michal; Fields, Gregg B (2018) Dissecting MMP P10' and P11' subsite sequence preferences, utilizing a positional scanning, combinatorial triple-helical peptide library. J Biol Chem 293:16661-16676
Pahwa, Sonia; Bhowmick, Manishabrata; Amar, Sabrina et al. (2018) Characterization and regulation of MT1-MMP cell surface-associated activity. Chem Biol Drug Des :
Van Doren, Steven R; Marcink, Tara C; Koppisetti, Rama K et al. (2017) Peripheral membrane associations of matrix metalloproteinases. Biochim Biophys Acta Mol Cell Res 1864:1964-1973
Amar, Sabrina; Smith, Lyndsay; Fields, Gregg B (2017) Matrix metalloproteinase collagenolysis in health and disease. Biochim Biophys Acta Mol Cell Res 1864:1940-1951
Prior, Stephen H; Byrne, Todd S; Tokmina-Roszyk, Dorota et al. (2016) Path to Collagenolysis: COLLAGEN V TRIPLE-HELIX MODEL BOUND PRODUCTIVELY AND IN ENCOUNTERS BY MATRIX METALLOPROTEINASE-12. J Biol Chem 291:7888-901
Singh, Warispreet; Fields, Gregg B; Christov, Christo Z et al. (2016) Importance of the Linker Region in Matrix Metalloproteinase-1 Domain Interactions. RSC Adv 6:23223-23232
Iyer, Rugmani Padmanabhan; de Castro BrĂ¡s, Lisandra E; Patterson, Nicolle L et al. (2016) Early matrix metalloproteinase-9 inhibition post-myocardial infarction worsens cardiac dysfunction by delaying inflammation resolution. J Mol Cell Cardiol 100:109-117
Fields, Gregg B; Stawikowski, Maciej J (2016) Imaging Matrix Metalloproteinase Activity Implicated in Breast Cancer Progression. Methods Mol Biol 1406:303-29

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